The balance between cell proliferation and differentiation is critical to the physiological function of the epithelium and is therefore well regulated, in part by transcription factors that control gene expression during these processes. Previous work has suggested that the ESE family of Ets transcription factors may play an important role in epithelial development and functional specialization. We have utilized skin keratinocytes as a model system to examine the biological role of ESE-2. Our preliminary studies characterizing the ESE-2 promoter have led to identification of a novel nuclear factor, KSF, that among all the cell types tested is restricted to keratinocytes and is important for the promoter activity. In order to gain insights into the biological function of ESE-2, we have begun a structure-function analysis of the protein. We show that recombinant ESE-2 isoforms can bind to known Ets-binding sites. However, the optimum DNA target for ESE-2 including the core motif and the flanking sequences remains to be determined. In order to define the in vivo role of ESE-2 and identify its target genes we have successfully targeted the ESE-2 gene in embryonic stem cell (ES) clones. Our studies indicate that a thorough analysis of the mechanism of action of ESE-2, its biological role and the identification of factors that regulate its expression should lead to a better understanding of epithelial cell differentiation process. Therefore, in the present proposal we seek to identify and characterize the regulatory factors that control the epithelium-specific expression of ESE-2, including the KSF protein (Aim 1), to perform a comprehensive structure-function analysis of all ESE-2 isoforms to identify their consensus binding sites and to characterize potential activation or repression domains that are critical for their specific roles in skin epidermis (Aim 2), and to employ an in vivo knock out model system to assess the biological role of ESE-2 during epithelial development and differentiation and to identify ESE-2 targets (Aim 3). Our studies will provide the tools to investigate the molecular mechanisms by which ESE-2 controls and shapes epithelial growth and differentiation and to identify important modulators of these processes. The studies proposed will thus provide fundamental insights into the biology of epithelial cells during both normal physiological conditions and abnormal pathological states. Such knowledge is vital to developing new therapies and cures for a wide array of diseases that afflict the epithelial cells including cancers.